1

Strawberry-tree, blackthorn and rose fruits: detailed characterization in nutrients 1

and phytochemicals with antioxidant properties 2
3
LILLIAN BARROS, ANA MARIA CARVALHO, JORGE S MORAIS, AND ISABEL C.F.R. 4
FERREIRA
*
5
6
CIMO/Escola Superior Agrria, Instituto Politcnico de Bragana, Campus de Santa 7
Apolnia, Apartado 1172, 5301-855 Bragana, Portugal. 8
9
10
* Author to whom correspondence should be addressed (e-mail: iferreira@ipb.pt 11
telephone +351-273-303219; fax +351-273-325405). 12
13
2
Abstract 14
The chemical composition and biological properties of three wild fruits (strawberry-tree 15
berries, sloes and dog rose hips) were evaluated, in order to valorise these products as 16
sources of nutrients and nutraceuticals. The analysed fruits contain very useful bioactive 17
phytochemicals such as phenolics, vitamins (ascorbic acid and tocopherols) and 18
carotenoids. All the samples proved to have antioxidant activity (measured by four 19
different in vitro assays) being more significant for rose fruits (EC
50
values lower than 20
90 g/mL). The combination of bioactive compounds and rich nutritional composition 21
(high contents in carbohydrates, low contents in fat with the precious contribution of 22
polyunsaturated fatty acids, precursors of omega-3 and omega-6 fatty acids) of the 23
studied wild fruits make them a very special food. 24
25
26
Keywords: Wild fruits; nutritional value; phytochemicals; bioactive properties 27
28
3
1. Introduction 29
30
The fruits of Arbutus unedo L. (Castroviejo, 1996) (Ericaceae), a species widely 31
distributed in the Mediterranean region and North Africa, are a red aggregate drupe 32
generally known as strawberry-tree berries. Their use is very popular in Portugal, 33
particularly made in a kind of strong brandy, the aguardente de medronho. In several 34
Portuguese regions (Trs-os-Montes, Alentejo and Algarve) the fruits eaten raw or made 35
in liqueurs, as well as, bark or roots decoctions, are used as anti-inflammatory, laxative, 36
carminative, digestive, odontalgic and cardiotonic (Novais, Santos, Mendes, & Pinto- 37
Gomes, 2004; Salgueiro, 2004; Carvalho, 2005; Camejo-Rodrigues, 2006). Some 38
literature report the traditional use of the leaves as a diuretic, urinary antiseptic, 39
antidiarrheal, astringent, depurative, against blenorrhagia, diabetes and as 40
antihypertensive (Ziyyat et al., 1997). Experimental investigations have already shown 41
that the aqueous extract of the plant exhibited antihypertensive (Ziyyat & Boussairi, 42
1998) and vasorelaxant (Ziyyat et al., 2002) activities. Sloes, the fruits of blackthorn, 43
Prunus spinosa L. (Castroviejo, 1998), a deciduous shrub native to Europe, have also 44
been used as astringent, diuretic and purgative (Lust, 1980). In the North-eastern 45
Portugal, the fruits are commonly eaten raw, prepared in jams or macerated with sugar, 46
honey and brandy to obtain a digestive and laxative liqueur, which is usually drunk after 47
copious meals (Novais et al., 2004; Salgueiro, 2004; Carvalho, 2005; Camejo- 48
Rodrigues, 2006). Moreover, rose hips, i.e. the pomaceous fruit of dog roses, Rosa 49
canina L., possess prophylactic and therapeutic activities against a wide range of 50
ailments, including the inflammatory disorders arthritis (Rein, Kharazmi, & Winther, 51
2004; Kharazmi, 2008), rheumatism, gout, and sciatica, for diseases with fever, for 52
colds and infectious diseases including influenza, against gastrointestinal disorders, to 53
4
aid digestion, prevention of inflammation of the gastric mucosa and gastric ulcer, for 54
gallstones, biliary complaints, as a laxative, for disorders of the kidney and the lower 55
urinary tract, as a diuretic, for dropsy and as an astringent (Orhan, Harteviolu, Kpeli, 56
& Yesilalada, 2007). In Portugal, rose hips from the Caninae DC. section (Castroviejo, 57
1998), a polymorphic group of scrambling rose species indigenous to Europe, northwest 58
Africa and western Asia, are also used in the treatment of colds, influenza, minor 59
infectious diseases, diarrhoea and as topical anti-inflammatory for muscular-skeletical 60
pathologies. Immature and ripened fruits of wild roses, mainly Rosa canina L and Rosa 61
corymbifera Borkh. (Castroviejo, 1998), were/are indistinctively applied in the folk 62
medicine of the Trs-os-Montes region. During late summer, the rose hips were 63
gathered and eaten raw as snacks or given raw to the children, who were taught how to 64
eat the outer fleshy hypantium and to spit out the dry single-seeded fruits (achenes) that 65
are embedded in a matrix of fine, but stiff, hairs (frequency of citation > 40%) 66
(Carvalho, 2005). In the surveyed area (north-eastern Portuguese region) wild fruits 67
were, sometime still are, commonly preserved and stored, for consumption during the 68
long and hard winters. Jams are prepared from arbutus berries, sloes, blackberries 69
(Rubus ulmifolius Schott.) and wild strawberries (Fragaria vesca L.). Despite the 70
brandy of arbutus berries, quite exceptional and much appreciated spirits are made from 71
sloes, blackberries and wild strawberries (frequency of citation > 55%) (Carvalho, 72
2005). 73
Epidemiological studies have consistently shown an inverse association between 74
consumption of vegetables and fruits and the risk of certain forms of cancer and 75
cardiovascular diseases (Bazzano, He, & Ogden, 2001). The protective effects have 76
been primarily attributed to antioxidants, such as Vitamin C, Vitamin E, -carotene and, 77
5
lately, to phenolic compounds (Soobrattee, Neergheen, Luximon-Ramma, Aruoma, & 78
Bahorun, 2005). There are only a few reports on antioxidant composition of strawberry- 79
tree (Spanish samples; Pallalauf, Rivas-Gonzalo, Castillo, Cano, & Pascual-Teresa, 80
2008) and rose fruits (German samples; Wenzig et al., 2008). Considering the 81
nutritional composition, the studies available in literature only report macronutrient 82
analysis (Demir & zcan, 2001; Marakolu, Arslan, zcan, & Hacseferoullar., 2005; 83
zcan & Hacseferoullar, 2007; Pallalauf et al., 2008). 84
Despite the high popularity of these wild fruits in Portugal, data regarding a complete 85
nutritional and phytochemical characterization are missing. The high nutritional quality 86
and bioactive compounds of these fruits are likely to be lost if not documented. Herein, 87
we intend to present a study of the chemical composition and antioxidant properties of 88
three Portuguese wild fruits (strawberry-tree, sloes and rose hips), in order to valorise 89
these products as sources of nutrients and nutraceuticals. Chemical analysis included 90
determination of proteins, fats, ash, and carbohydrates, and individual profiles in sugars 91
and fatty acids by chromatographic techniques. Phytochemicals such as phenolics, 92
flavonoids, vitamins (tocopherols and ascorbic acid), and carotenoids were also 93
determined. 94
95
2. Materials and methods 96
97
2.1. Samples 98
The fruits of Arbutus unedo, Prunus spinosa and Rosa canina sl. were gathered in the 99
Natural Park of Montesinho territory, in Trs-os-Montes, North-eastern Portugal, 100
according to the folk uses of each species (Table 1), especially those concerning fruit 101
6
ripening stage and the most suitable gathering period and practices. Strawberry-tree 102
berries were collected fully ripened in November 2008; well matured sloes and rose 103
hips were gathered in late September 2008. Morphological key characters from the 104
Flora Iberica (Castroviejo, 1996; Castroviejo, 1998) were used for plant identification. 105
The fruits with seeds were lyophilized (Ly-8-FM-ULE, Snijders, HOLLAND) and kept 106
in the best conditions for subsequent use. 107
108
2.2. Standards and Reagents 109
Acetonitrile 99.9%, n-hexane 95% and ethyl acetate 99.8% were of HPLC grade from 110
Lab-Scan (Lisbon, Portugal). All the other solvents were of analytical grade purity: 111
methanol and diethyl ether were supplied by Lab-Scan (Lisbon, Portugal), while toluene 112
and sulphuric acid were supplied by Sigma Chemical Co. (St. Louis, MO, USA). The 113
fatty acids methyl ester (FAME) reference standard mixture 37 (fatty acids C4 to C24; 114
(standard 47885-U) was purchased from Sigma (St. Louis, MO, USA), as also other 115
individual fatty acid isomers, tocopherol standards (, , and ), and the standards 116
used in the antioxidant activity assays: trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- 117
carboxylic acid), gallic acid and (+)-catechin. Racemic Tocol, 50 mg/ml, was purchased 118
from Matreya (PA, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from 119
Alfa Aesar (Ward Hill, MA, USA). All other chemicals were obtained from Sigma 120
Chemical Co. (St. Louis, MO, USA). Water was treated in a Milli-Q water purification 121
system (TGI Pure Water Systems, USA). 122
123
2.3. Nutrients composition 124
7
Macronutrients. The samples were analysed for chemical composition (moisture, 125
protein, fat, carbohydrates and ash) using the AOAC procedures (AOAC, 1995). The 126
crude protein content (N 6.25) of the samples was estimated by the macro-Kjeldahl 127
method; the crude fat was determined by extracting a known weight of powdered 128
sample with petroleum ether, using a Soxhlet apparatus; the ash content was determined 129
by incineration at 60015 C. Total carbohydrates were calculated by difference: Total 130
carbohydrates = 100 (g moisture + g protein + g fat + g ash). Total energy was 131
calculated according to the following equations: Energy (kcal) = 4 (g protein +g 132
carbohydrate) + 9 (g lipid). 133
134
Fatty Acids. Fatty acids were determined by gas-liquid chromatography with flame 135
ionization detection (GC-FID)/capillary column as described previously by the authors 136
(Barros, Venturini, Baptista, Estevinho, & Ferreira, 2008), and after the following trans- 137
esterification procedure: fatty acids (obtained after Soxhlet extraction) were methylated 138
with 5 mL of methanol:sulphuric acid:toluene 2:1:1 (v:v), during at least 12 h in a bath 139
at 50 C and 160 rpm; then 3 mL of deionised water were added, to obtain phase 140
separation; the FAME were recovered with 3 mL of diethyl ether by shaking in vortex , 141
and the upper phase was passed through a micro-column of sodium sulphate anhydrous, 142
in order to eliminate the water; the sample was recovered in a vial with Teflon, and 143
before injection the sample was filtered with 0.2 m nylon filter from Milipore. The 144
fatty acid profile was analyzed with a DANI model GC 1000 instrument equipped with 145
a split/splitless injector, a flame ionization detector (FID) and a Macherey-Nagel 146
column (30 m x 0.32 mm ID x 0.25 m d
f
). The oven temperature program was as 147
follows: the initial temperature of the column was 50 C, held for 2 min, then a 148
8
10C/min ramp to 240 C and held for 11 min. The carrier gas (hydrogen) flow-rate was 149
4.0 mL/min (0.61 bar), measured at 50 C. Split injection (1:40) was carried out at 250 150
C. For each analysis 1 L of the sample was injected in GC. Fatty acid identification 151
was made by comparing the relative retention times of FAME peaks from samples with 152
standards. The results were recorded and processed using CSW 1.7 software (DataApex 153
1.7) and expressed in relative percentage of each fatty acid. 154
155
Sugars. Free sugars were determined by high performance liquid chromatography 156
coupled to a refraction index detector (HPLC-RI) as described by Barros et al. (2008) 157
with some modifications. Dried sample powder (1.0 g) was spiked with the melezitose 158
as internal standard (IS, 5 mg/ml), and was extracted with 40 mL of 80% aqueous 159
ethanol at 80 C for 30 min. The resulting suspension was centrifuged (Centorion 160
K24OR- 2003 refrigerated centrifuge) at 15,000 g for 10 min. The supernatant was 161
concentrated at 60 C under reduced pressure and defatted three times with 10 mL of 162
ethyl ether, successively. After concentration at 40 C, the solid residues were dissolved 163
in water to a final volume of 5 mL. Soluble sugars were determined by using HPLC 164
(Knauer, Smartline system) at 35 C. The HPLC system was equipped with a Knauer 165
Smartline 2300 RI detector and with a Eurospher 100-5 NH
2
column (4.6 x 250 mm, 5 166
mm, Knauer). The mobile phase was acetonitrile/deionized water, 7:3 (v/v) at a flow 167
rate of 1 mL/min. The results are expressed in g/100 g of dry weight, calculated by 168
internal normalization of the chromatographic peak area. Sugar identification was made 169
by comparing the relative retention times of sample peaks with standards. The sugar 170
standards used for identification were purchased from Sigma Chemical Co. (St. Louis, 171
USA): L(+)-arabinose, D(-)-fructose, L-fucose, D(+)-galactose, D(+)-glucose 172
9
anhydrous, lactose 1-hydrate, maltose 1-hydrate, maltulose monohydrate, D(+)- 173
mannitol, D(+)-mannose, D(+)-melezitose, D(+)-melibiose monohydrate, D(+)- 174
raffinose pentahydrate, L(+)-rhamnose monohydrate, D(+)-sucrose, D(+)-trehalose, 175
D(+)- turanose and D(+)-xylose. 176
177
2.4. Phytochemicals composition 178
Tocopherols. Tocopherols content was determined following a procedure previously 179
optimized and described by Barros, Heleno, Carvalho, & Ferreira (in press). BHT 180
solution in hexane (10 mg/mL; 100 L) and IS solution in hexane (tocol; 50 g/mL; 400 181
L) were added to the sample prior to the extraction procedure. The samples (~500 mg) 182
were homogenized with methanol (4 mL) by vortex mixing (1 min). Subsequently, 183
hexane (4 mL) was added and again vortex mixed for 1 min. After that, saturated NaCl 184
aqueous solution (2 mL) was added, the mixture was homogenized (1 min), centrifuged 185
(5 min, 4000g) and the clear upper layer was carefully transferred to a vial. The sample 186
was re-extracted twice with hexane. The combined extracts were taken to dryness under 187
a nitrogen stream, redissolved in 2 mL of n-hexane, dehydrated with anhydrous sodium 188
sulphate, filtered through a 0.22 m disposable LC filter disk, transferred into a dark 189
injection vial and analysed by HPLC. The HPLC equipment consisted of an integrated 190
system with a Smartline pump 1000 (Knauer, Germany), a degasser system Smartline 191
manager 5000, an AS-2057 auto-sampler and a 2500 UV detector at 295 nm (Knauer, 192
Germany) connected in series with a FP-2020 fluorescence detector (Jasco, Japan) 193
programmed for excitation at 290 nm and emission at 330 nm. Data were analysed 194
using Clarity 2.4 Software (DataApex). The chromatographic separation was achieved 195
with a Polyamide II (250 x 4.6 mm) normal-phase column from YMC Waters (Japan) 196
10
operating at 30C (7971 R Grace oven). The mobile phase used was a mixture of n- 197
hexane and ethyl acetate (70:30, v/v) at a flow rate of 1 ml/min, and the injection 198
volume was 20 L. The compounds were identified by chromatographic comparisons 199
with authentic standards. Quantification was based on the fluorescence signal response, 200
using the internal standard method. Tocopherol contents in the samples are expressed in 201
g per g of dry sample. 202
203
Ascorbic acid. Ascorbic acid was determined according to the method of Klein and 204
Perry (1982). A fine powder (20 mesh) of sample (150 mg) was extracted with 205
metaphosphoric acid (1%, 10 mL) for 45 min at room temperature and filtered through 206
Whatman N 4 filter paper. The filtrate (1 mL) was mixed with 2,6-dichloroindophenol 207
(9 mL) and the absorbance was measured within 30 min at 515 nm against a blank 208
(Analytikijena 200-2004 spectrophotometer). Content of ascorbic acid was calculated 209
on the basis of the calibration curve of authentic L-ascorbic acid (0.006-0.1 mg/mL; 210
y = 3.0062x + 0.007; R
2
= 0.9999), and the results were expressed as g of ascorbic 211
acid/g of dry weight. 212
213
Carotenoids. -Carotene and lycopene were determined according to the method of 214
Nagata and Yamashita (1992). A fine dried powder (150 mg) was vigorously shaken 215
with 10 mL of acetonehexane mixture (4:6) for 1 min and filtered through Whatman 216
No. 4 filter paper. The absorbance of the filtrate was measured at 453, 505, 645 and 663 217
nm. Contents of -carotene and lycopene were calculated according to the following 218
equations: lycopene (mg/100 mL) = - 0.0458 A
663
+ 0.204 A
645
+ 0.372 A
505
- 219
11
0.0806 A
453
; -carotene (mg/100 mL) = 0.216 A
663
1.220 A
645
- 0.304 A
505
+ 220
0.452 A
453
. The results were expressed as g of carotenoid/g of dry weight. 221
222
Phenolics. A fine dried powder (20 mesh; ~1g) was extracted by stirring with 50 mL of 223
methanol at 25 C at 150 rpm for 12 h and filtered through Whatman No. 4 paper. The 224
residue was then extracted with one additional 50 mL portion of methanol. The 225
combined methanolic extracts were evaporated at 35C under reduced pressure (rotary 226
evaporator Bchi R-210), re-dissolved in methanol at a concentration of 10 mg/mL, and 227
stored at 4 C for further use. 228
Total phenolics were estimated based on procedures described by Wolfe, Wu, & Liu 229
(2003) with some modifications. An aliquot of the extract solution (1 mL) was mixed 230
with Folin-Ciocalteu reagent (5 mL, previously diluted with water 1:10 v/v) and sodium 231
carbonate (75 g/L, 4 mL). The tubes were vortexed for 15 s and allowed to stand for 232
30 min at 40 C for colour development. Absorbance was then measured at 765 nm. 233
Gallic acid was used to calculate the standard curve (0.05-0.8 mM; y = 1.9799x + 234
0.0299; R
2
= 0.9997), and the results were expressed as mg of gallic acid equivalents 235
(GAEs) per g of extract. 236
Total flavonoid content was determined using the method of Jia Tang, & Wu (1999), 237
with some modifications. An aliquot (0.5 mL) of the extract solution was mixed with 238
distilled water (2 mL) and subsequently with NaNO
2
solution (5%, 0.15 mL). After 6 239
min, AlCl
3
solution (10%, 0.15 mL) was added and allowed to stand further 6 min, 240
thereafter, NaOH solution (4%, 2 mL) was added to the mixture. Immediately, distilled 241
water was added to bring the final volume to 5 mL. Then the mixture was properly 242
mixed and allowed to stand for 15 min. The intensity of pink colour was measured at 243
12
510 nm. (+)-Catechin was used to calculate the standard curve (0.0156-1.0 mM; 244
y = 0.9186x - 0.0003; R
2
= 0.9999) and the results were expressed as mg of (+)- 245
chatequin equivalents (CEs) per g of extract. 246
247
2.5. In vitro evaluation of the antioxidant properties 248
Chemical assays already described by the authors in previous studies (Barros et al., in 249
press), were applied to evaluate the antioxidant activity of all the samples. Different 250
concentration of the extracts (10 mg/mL to 0.05 mg/mL) were used to find EC
50
values. 251
DPPH radical-scavenging activity. This methodology was performed using an ELX800 252
Microplate Reader (Bio-Tek Instruments, Inc). The reaction mixture in each one of the 253
96-wells consisted of one of the different concentrations of the extracts (30 L) and 254
aqueous methanolic solution (80:20 v/v, 270 L) containing DPPH radicals (6x10
-5
255
mol/L). The mixture was left to stand for 60 min in the dark. The reduction of the DPPH 256
radical was determined by measuring the absorption at 515 nm. The radical scavenging 257
activity (RSA) was calculated as a percentage of DPPH discolouration using the 258
equation: % RSA = [(A
DPPH
-A
S
)/A
DPPH
] 100, where A
S
is the absorbance of the 259
solution when the sample extract has been added at a particular level, and A
DPPH
is the 260
absorbance of the DPPH solution. The extract concentration providing 50% of radicals 261
scavenging activity (EC
50
) was calculated from the graph of RSA percentage against 262
extract concentration. Trolox was used as standard. 263
264
Reducing power. This methodology was performed using the Microplate Reader 265
described above. The different concentrations of the extracts (0.5 mL) were mixed with 266
sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and potassium ferricyanide (1% 267
13
w/v, 0.5 mL). The mixture was incubated at 50 C for 20 min, and trichloroacetic acid 268
(10% w/v, 0.5 mL) was added. The mixture (0.8 mL) was poured in the 48-wells, as 269
also deionised water (0.8 mL) and ferric chloride (0.1% w/v, 0.16 mL), and the 270
absorbance was measured at 690 nm. The extract concentration providing 0.5 of 271
absorbance (EC
50
) was calculated from the graph of absorbance at 690 nm against 272
extract concentration. Trolox was used as standard. 273
274
Inhibition of -carotene bleaching. A solution of -carotene was prepared by dissolving 275
-carotene (2 mg) in chloroform (10 mL). Two millilitres of this solution were pipetted 276
into a round-bottom flask. After the chloroform was removed at 40C under vacuum, 277
linoleic acid (40 mg), Tween 80 emulsifier (400 mg), and distilled water (100 mL) were 278
added to the flask with vigorous shaking. Aliquots (4.8 mL) of this emulsion were 279
transferred into different test tubes containing different concentrations of the extracts 280
(0.2 mL). The tubes were shaken and incubated at 50C in a water bath. As soon as the 281
emulsion was added to each tube, the zero time absorbance was measured at 470 nm 282
using a spectrophotometer. A blank, devoid of -carotene, was prepared for background 283
subtraction. -Carotene bleaching inhibition was calculated using the following 284
equation: (-carotene content after 2h of assay/initial -carotene content) 100. The 285
extract concentration providing 50% antioxidant activity (EC
50
) was calculated by 286
interpolation from the graph of -carotene bleaching inhibition percentage against 287
extract concentration. Trolox was used as standard. 288
289
Inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS). 290
Brains were obtained from pig (Sus scrofa) of body weight ~150 Kg, dissected and 291
14
homogenized with a Polytron in ice-cold TrisHCl buffer (20 mM, pH 7.4) to produce a 292
1:2 (w/v) brain tissue homogenate which was centrifuged at 3000g for 10 min. An 293
aliquot (0.1 mL) of the supernatant was incubated with the different concentrations of 294
the extracts (0.2 mL) in the presence of FeSO
4
(10 M; 0.1 ml) and ascorbic acid (0.1 295
mM; 0.1 ml) at 37C for 1 h. The reaction was stopped by the addition of trichloroacetic 296
acid (28% w/v, 0.5 mL), followed by thiobarbituric acid (TBA, 2%, w/v, 0.38 mL), and 297
the mixture was then heated at 80 C for 20 min. After centrifugation at 3000g for 10 298
min to remove the precipitated protein, the colour intensity of the malondialdehyde 299
(MDA)-TBA complex in the supernatant was measured by its absorbance at 532 nm. 300
The inhibition ratio (%) was calculated using the following formula: Inhibition ratio 301
(%) = [(A B)/A] x 100%, where A and B were the absorbance of the control and the 302
compound solution, respectively. The extract concentration providing 50% lipid 303
peroxidation inhibition (EC
50
) was calculated from the graph of TBARS inhibition 304
percentage against extract concentration. Trolox was used as standard. 305
306
2.6. Statistical analysis 307
For each one of the species three samples were analysed and also all the assays were 308
carried out in triplicate. The results are expressed as mean values and standard deviation 309
(SD) or standard errors (SE). The results were analyzed using one-way analysis of 310
variance (ANOVA) followed by Tukeys HSD Test with = 0.05. This treatment was 311
carried out using SPSS v. 16.0 program. 312
313
15
3. Results and discussion 314
We performed a nutritional and pytochemical characterization of three wild Portuguese 315
fruits: Strawberry-tree (A. unedo), blackthorn (P. spinosa) and rose (R. canina sl.) fruits. 316
The ethnobotanical survey conducted in the North-eastern Portugal (Carvalho, 2005) 317
has reported that in former times the studied wild fruits were often eaten as snacks 318
during the working day in the crop fields, in the meadows while the cattle was grazing 319
or in the forest. Moreover the mothers forced their children to eat them raw when fully 320
ripened because they were convinced it was healthy. As several studies have also 321
documented (Gonzlez-Tejero et al., 2008; Hadjichambis et al., 2008) the wild fruits 322
resulting products such as marmalades, spirits and infusions were also deliberately 323
consumed for their preventative or curative properties and are considered medicinal 324
foods(Carvalho, 2005; Gonzlez-Tejero et al., 2008). 325
326
3.1. Nutrients composition 327
The results of the nutrients composition and estimated energetic value (expressed on dry 328
weight basis) obtained for the wild fruits are shown in Table 1. Blackthorn fruits 329
revealed the highest moisture content (60.86 g/100 g), a similar value to the described 330
by Marakolu et al. (2005) for Turkish samples (69.37%). Portuguese strawberry-tree 331
fruits revealed a slightly higher moisture content (59.70 g/100 g) than Turkish fruits 332
(53.72%) (zcan & Hacseferoullar, 2007). Otherwise, Portuguese rose fruits showed 333
lowest moisture contents (48.68 g/100 g) than Turkish samples (69.52%) 334
(Hacseferoullar, zcan, Sonmete, & zbek, 2005). 335
Carbohydrates, calculated by difference, were the most abundant macronutrients and 336
were higher than 88.5%. In fact, these fruits are rich in different carbohydrates, either 337
16
monosaccharides or polysaccharides such as cellulose and starch (Demir & zcan, 338
2001; zcan & Hacseferoullar, 2007). 339
The results show that the consumption of these fruits as snacks was appropriate for the 340
particular purpose of satisfying hunger in view of their carbohydrates content. In fact 341
some informants highlighted the importance of wild plants in local daily diet 342
specifically during famine periods, such as those occurring when the Spanish Civil War 343
and the Second World War (Carvalho, 2005). 344
Protein was found in low levels and varied between 2.72 g/100 g in rose fruits and 3.09 345
g/100 g in strawberry-tree fruits. The Turkish samples of rose (6.71 to 8.44%, Demir & 346
zcan, 2001), blackthorn (3.4%, Marakolu et al., 2005) and strawberry-tree (3.36%, 347
zcan & Hacseferoullar, 2007) showed highest proteins levels. Fat was the 348
macronutrient less abundant being lower than 2% and also lower than those found in 349
Turkish fruits: 2.1% (zcan & Hacseferoullar, 2007), 2.06% (Marakolu et al., 2005) 350
and 1.2 to 1.6% (Demir & zcan, 2001) for strawberry-tree, blackthorn and rose, 351
respectively. On the basis of the proximate analysis, it can be calculated that a dry 352
portion of 100 g of these fruits assures, on average, 394 Kcal. The highest values are 353
guaranteed by strawberry-tree fruits, while blackthorn fruits give the lowest energy 354
contribution (Table 1). Ash content fall between proteins and fat contents, being more 355
abundant in blackthorn fruits (6.65 g/100 g), a much higher value than the one found in 356
Turkish samples (2.72%, Marakolu et al., 2005). The lowest values were found in 357
strawberry-tree fruits (1.71 g/100 g), which was lower than the value reported in the 358
Turkish samples (2.82%, zcan & Hacseferoullar, 2007). 359
Despite some similarities in the composition of Portuguese and Turkish samples, it is 360
known that differences in chemical properties of fruits having about the same size were 361
17
probably due to environmental conditions in conjunction with the analytical methods 362
used. In addition, moisture, crude protein, fibre, fat and ash contents of fruits are 363
affected chiefly by variety and growth conditions (Hacseferoullar et al., 2005). 364
365
The results for fatty acid composition, total saturated fatty acids (SFA), 366
monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) of the 367
studied wild fruits are shown in Table 2.The major fatty acids found in strawberry-tree 368
and rose fruits were -linolenic acid (C18:3) and linoleic acid (C18:2), contributing to 369
the prevalence of PUFA in these samples. -Linolenic and linoleic acids are essential 370
fatty acids as they cannot be synthesised by the human organism, due to the lack of 371
desaturase enzymes required for their production. They must be obtained by the diet and 372
originate the omega-3 and omega-6 fatty acids, respectively (Voet & Voet, 2004). These 373
omega fatty acids are the biosynthetic precursor of eicosanoids, meaning that their 374
intake concentrations will strongly influence eicosanoids production, and, therefore, the 375
organisms metabolic functions. Furthermore, they can also decrease the total amount of 376
fat in blood (cholesterol), reducing the risk of cardiovascular diseases (Voet & Voet, 377
2004; Kanu et al., 2007). In blackthorn fruits, PUFA predominated over MUFA due to 378
the abundance of oleic acid (C18:1). These fruits also present high levels of linoleic acid 379
but significant lower amounts of -linolenic acid than the other two wild fruits. In all 380
the cases UFA predominate over SFA, ranging from 80 to 84%, being palmitic acid the 381
main SFA found. Twenty four fatty acids were identified and quantified. As far as we 382
know, nothing has been reported on fatty acid composition of strawberry-tree and 383
blackthorn fruits. Nevertheless, Wenzig et al. (2008) also reported palmitic, linoleic and 384
18
-linolenic acid as the main free fatty acids present in rose hip extracts from German 385
fruits. 386
387
In what concerns sugar composition (Table 3) the wild fruits presented fructose, 388
glucose and sucrose as main sugars. The present study describes for the first time the 389
sugars composition in these wild fruits. For strawberry-tree (24.21 g/100 g) and rose 390
fruits (12.89 g/100 g) fructose was the most abundant sugar, while glucose 391
predominates in blackthorn samples (29.84 g/100 g). Strawberry-tree fruits revealed the 392
highest total sugars content, and highest levels of fructose and sucrose, which is in 393
agreement with its sweet taste. Otherwise, rose fruits showed the lowest levels in total 394
sugars (26.90 g/100 g). 395
Sugars contents (Table 3) are a significant part of carbohydrates (Table 1), but other 396
carbohydrates are also present in these fruits such as polysaccharides (eg. cellulose and 397
starch) (Demir & zcan, 2001; zcan & Hacseferoullar, 2007). 398
399
Overall, strawberry-tree fruits revealed the highest energetic value, but with the highest 400
carbohydrates and proteins content. Concerning sugar composition it presents the 401
highest percentage of fructose, which is one of the important dietary monosaccharides 402
and it is known to be the sweetest of all naturally occurring carbohydrates (Hanover & 403
White, 1993). Strawberry-tree (A. unedo) fruits are considered an excellent edible to 404
make delicious and nourishing jams and conserves due to its sweetness and delicate 405
pleasant flavour. Rose fruits presented the highest PUFA, which may be relevant since 406
-linolenic and linoleic acids are precursors of omega-3 and omega-6 fatty acids often 407
related to an increase in HDL cholesterol and decrease in LDL cholesterol, 408
19
triacylglycerol, lipid oxidation, and LDL susceptibility to oxidation (Voet & Voet, 409
2004; Kanu et al., 2007). Although the edible use of rose fruits is less frequent in 410
Portugal, than arbutus berries and sloes, even than its medicinal use, rose hips are 411
considered an excellent ingredient for making delicious jams, syrups and herbal teas, in 412
several European regions (Tardo, Pardo de Santayana, & Morales, 2006; Gonzlez- 413
Tejero et al., 2008; Hadjichambis et al., 2008). 414
415
3.2. Phytochemicals composition 416
Vitamins (tocopherols and ascorbic acid) and carotenoids contents in the wild fruits 417
were determined and the results are given in Table 4. 418
The values obtained in the analysis of the samples point to the existence of differences 419
in what concerns tocopherols composition. -Tocopherol was the major compound in 420
all the fruits, and -tocopherol was only detected in blackthorn fruits. Strawberry-tree 421
fruits presented the highest content of tocopherols (23.46 mg/ 100 g of dry weight; 422
Table 4, Figure 1) while rose fruits revealed the lowest content (8.33 mg/100 g). Even 423
if considering the fact that fruits in general have very low content of this vitamin, 424
characteristic of fat-rich foods, the content was not negligible. Some authors published 425
tocopherols determination in Spanish strawberry-tree fruits, but reporting only the 426
presence of -tocopherol (Pallalauf et al., 2008). In our study, we used a different 427
extraction methodology with the introduction of an antioxidant protector to minimize 428
tocopherols loss, and we used fluorescence detection, which is more sensitive than UV 429
detection, allowing us to quantify other vitamers such as , , and -tocopherols. 430
Regarding tocopherols in rose and blackthorn fruits, as far as we know, nothing is 431
described in literature. The health benefits of tocopherol as a bioactive compound are 432
20
well documented. -Tocopherol, the principal form of vitamin E, is a lipid-soluble 433
antioxidant and it functions as a chain-breaking antioxidant for lipid peroxidation (LP) 434
in cell membranes and also as a scavenger of ROS (Reactive Oxygen Species) such as 435
singlet oxygen. It is considered to serve as the first line of defence against LP, and it 436
protects PUFAs (polyunsaturated fatty acids) in cell membranes from free radical attack 437
through its scavenging activity in biomembranes at early stages of LP (Kanu et al., 438
2007). 439
Ascorbic acid was the most abundant vitamin in blackthorn and rose fruits, and 440
particularly for the latter sample it presented a very high level (68.04 mg/100 g dry 441
weight; Table 4). In fact, rose fruits have been used as a source of vitamin C in tea and 442
other products for many years (Krharazmi, 2008). Weinzig et al. (2008) reported the 443
quantification of ascorbic acid in German rose fruits, but the values are expressed in % 444
of dry extract and not of dry sample, which can not be comparable. Also, the 445
quantification of vitamin C in Spanish strawberry-tree fruits was reported (Pallalauf et 446
al., 2008), being the values described lower than the contents found in our Portuguese 447
fruits. Levels of vitamin C could be comparable to those in fruits like peaches, apples or 448
plums. Regarding blackthorn fruits, as far as we know, nothing is described in literature. 449
Carotenoids are widespread pigments in plants, being involved in photosynthesis and 450
photoprotection (Hodisan, Socaciu, Ropan, & Neamtu, 1997); -carotene was found in 451
small amounts in all the fruits (lower than 1.3 mg/100 g dry weight) and lycopene was 452
only detected in rose fruits. This is in agreement with the results of rose fruits from 453
Romania reported by Hodisan et al. (1997). The antioxidant properties of carotenoids 454
have been suggested as being the main responsible for their beneficial effects (Rao & 455
Rao, 2007). Particularly, -carotene has been found to be inversely associated with 456
21
cancer risk in epidemiologic studies and showed promising results in laboratory assays. 457
Also, the role of lycopene in the prevention of chronic diseases has been evaluated in 458
epidemiological studies as well as in tissue culture experiments using human cancer cell 459
lines, animal studies and also human clinical trials (Rao & Rao, 2007). 460
461
Table 5 presents the yields of the methanolic extraction and the phenolics and 462
flavonoids concentrations obtained in the wild fruits extracts. It was not observed any 463
correlation between the extraction yield and the phenolics contents. Phenolics were the 464
major antioxidant components (83.40-143.17 mg/g of extract) and rose fruits revealed 465
the highest content in phenolics. The amount found in our sample was higher than the 466
ones found in German rose fruits (82.2 to 133.1 mg/g of extract) (Wenzig et al., 2008). 467
Phenolic compounds exhibit a wide range of biological effects including antibacterial, 468
anti-inflammatory, antiallergic, hepatoprotective, antithrombotic, antiviral, 469
anticarcinogenic and vasodilatory actions (Soobrattee et al., 2005); many of these 470
biological functions have been attributed to their free radical scavenging and antioxidant 471
activity. Flavonoids are the most common and widely distributed group of plant 472
phenolics, and have been shown to be highly effective scavengers of most types of 473
oxidizing molecules, including singlet oxygen and various free radicals, which are 474
possibly involved in DNA damage and tumour promotion (Marchand, 2002). The 475
flavonoids content ranged from 8.68 to 34.99 mg/g of extract. 476
477
3.3. In vitro evaluation of the antioxidant properties 478
The antioxidant properties were evaluated using the whole extract, taking advantage of 479
the complex mixture of phytochemicals with potential additive and synergistic effects 480
22
(Liu, 2004). Several in vitro chemical and biochemical assays using animal cells were 481
performed: reducing power (measuring the conversion of a Fe
3+
/ferricyanide complex to 482
the ferrous form), scavenging activity on DPPH radicals (measuring the decrease in 483
DPPH radical absorption after exposure to radical scavengers), inhibition of -carotene 484
bleaching (by neutralizing the linoleate-free radical and other free radicals formed in the 485
system which attack the highly unsaturated -carotene models), and inhibition of lipid 486
peroxidation in brain tissue (measured by the colour intensity of MDA-TBA complex). 487
All the samples proved to have antioxidant activity (table 5) being more significant for 488
rose fruits (lowest EC
50
values). Blackthorn fruits presented the lowest antioxidant 489
properties (highest EC
50
values) which are compatible to its lower phenolics and 490
flavonoids content. 491
492
Overall, strawberry-trees revealed the highest contents in carbohydrates, proteins, 493
sugars, tocopherols and flavonoids, while rose fruits showed the highest content in 494
PUFA, ascorbic acid, carotenoids and phenolics, and the highest antioxidant properties. 495
The combination of the useful phytochemicals found in the analysed wild fruits and 496
their nutritional composition (particularly high contents in carbohydrates and low 497
contents in fat with the precious contribution of polyunsaturated fatty acids precursor of 498
omega-3 and omega-6 fatty acids) make them very special. This study contributes not 499
only to a better knowledge of these wild fruits but also to their valorisation. 500
The contribution of wild food plants to the total dietary intake has not yet been 501
estimated, but it is generally considered as being not very significant. However, in the 502
past, the ingestion of these fruits did provide important minor nutrients (such as 503
vitamins and essential fatty acids that were not present in daily meals mostly based on 504
23
bread and potatoes (Carvalho, 2005). Furthermore, the report of the radical scavenging 505
activity and lipid peroxidation inhibition capacity of these species from North-eastern 506
Portugal could help in the explanation of their uses in folk medicine against several 507
chronic diseases known to be related to the production of ROS and oxidative stress. 508
509
Acknowledgement 510
The authors are grateful to the Foundation for Science and Technology (Portugal) for 511
financial support to the research centre CIMO and L. Barros grant 512
(SFRH/BPD/4609/2008). 513
514
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619
28
Table 1. Moisture (g/100 g of fresh weight), nutrients composition (g/100 g of dry 620
weight) and energetic value (Kcal/100 g of dry weight) of the wild fruits (mean SD; 621
n=3). In each row, different letters mean significant differences (p<0.05). 622
623
Strawberry-Tree Blackthorn Rose
Moisture
59.70 2.67 b 60.86 1.69 a 48.68 0.91 c
Carbohydrates
93.83 0.41 a 88.51 2.24 b 93.16 0.18 a
Proteins
3.09 0.08 a 2.86 0.03 b 2.72 0.05 c
Fat
1.37 0.40 b 1.98 0.32 a 0.65 0.04 c
Ash
1.71 0.09 b 6.65 2.03 a 3.47 0.20 b
Energy
399.99 1.17 a 383.27 7.09 b 398.37 0.92 b
29
Table 2. Fatty acids composition of the wild fruits. The results are expressed as mean 624
SD (n=3). In each column different letters mean significant differences (p<0.05). 625
626

Strawberry-Tree Blackthorn Rose
C6:0
0.04 0.00 nd 0.05 0.00
C8:0
0.04 0.00 0.01 0.00 0.05 0.00
C10:0
0.04 0.00 0.02 0.00 0.09 0.00
C12:0
0.65 0.05 0.10 0.00 0.58 0.02
C13:0
0.07 0.00 nd 0.07 0.00
C14:0
1.34 0.15 0.09 0.00 0.36 0.02
C14:1
nd 0.03 0.00 0.02 0.00
C15:0
0.10 0.00 0.03 0.00 0.11 0.01
C16:0
8.20 0.25 6.50 0.29 6.72 0.03
C16:1
0.11 0.01 0.67 0.04 1.33 0.05
C17:0
0.30 0.01 0.11 0.01 0.25 0.01
C17:1c
nd 0.10 0.00 nd
C18:0
4.00 0.17 2.51 0.15 2.41 0.13
C18:1n9c
21.01 0.04 57.58 0.39 14.43 0.28
C18:2n6c
21.50 0.06 23.57 0.37 39.51 0.69
C18:3n3
36.51 0.64 2.79 0.20 26.33 0.11
C20:0
0.61 0.05 0.56 0.04 1.00 0.04
C20:1c
0.27 0.02 0.06 0.00 0.38 0.03
C20:2c
0.16 0.01 nd 0.40 0.04
C20:3n3+C21:0
0.12 0.01 0.03 0.00 0.12 0.01
C22:0
0.81 0.03 0.32 0.06 0.66 0.06
C23:0
2.68 0.10 4.42 0.02 1.75 0.04
C22:6n3
nd nd 2.07 0.01
C24:0
1.45 0.17 0.48 0.02 1.31 0.03
Total SFA
20.32 0.57 a 15.16 0.16 b 15.40 0.42 b
Total MUFA
21.39 0.03 b 58.45 0.34 a 16.16 0.02 c
Total PUFA
58.28 0.54 b 26.40 0.17 c 68.44 0.44 a
nd- not detected 627
30
Table 3. Sugars composition (g/100 g of dry weight) of the wild fruits (mean SD; 628
n=3). In each row, different letters mean significant differences (p<0.05). 629
630
631
632
633
634
635
636
637
638
Strawberry-Tree Blackthorn Rose
Fructose 24.21 1.46 a 6.95 0.46 c 12.89 0.94 b
Glucose 12.14 0.26 b 29.84 1.49 a 12.17 0.95 b
Sucrose 4.20 0.04 a 0.27 0.03 c 1.83 0.27 b
Total sugars 40.55 1.62 a 37.06 1.92 a 26.90 2.16 b
31
Table 4. Tocopherols, ascorbic acid and carotenoids composition (mg/100 g dry 639
weight) of the wild fruits. The results are expressed as mean SD (n=3). In each row 640
different letters mean significant differences (p<0.05). 641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
nd- not detected. 662
Strawberry-Tree Blackthorn Rose
-tocopherol 21.98 0.18 a 7.18 0.34 b 7.05 0.28 b
-tocopherol 0.44 0.02 a 0.06 0.01 c 0.19 0.01 b

-tocopherol 1.03 0.06 b 1.91 0.28 a 1.09 0.06 b
-tocopherol nd 0.10 0.01 nd
Total tocopherols 23.46 0.26 a 9.25 0.64 b 8.33 0.34 b
Ascorbic acid 15.07 0.77 b 15.69 0.53 b 68.04 1.11 a
-carotene 1.07 0.09 b 0.78 0.01 c 1.29 0.26 a
Lycopene nd nd 0.51 0.08
32
Table 5. Extraction yields, phenolics, flavonoids and antioxidant activity EC
50
values of the wild fruits. The results are expressed as mean SD 663
(n=3). In each column different letters mean significant differences (p<0.05). 664
665
Antioxidant properties (EC
50
values; g/ml)
DPPH scavenging
activity
Reducing
power
-carotene bleaching
inhibition
TBARS
inhibition
447.92 0.81 b 410.80 0.93 b 774.99 0.86 b 94.27 1.21 b
597.50 0.43 a 607.44 0.38 a 986.90 0.91 a 153.86 1.98 a

428.84 0.71 b 171.23 0.79 c 396.06 3.84 c 87.20 2.17 c
666



33
667
668
669
670
671
672
673
674
675
676
Figure 1. HPLC fluorescence chromatogram of Arbutus unedo (strawberry-tree) 677
fruits. Peaks: 1- -tocopherol; 2- BHT; 3- -tocopherol; 4- -tocopherol; 5-IS 678
(tocol). 679
680
Voltage (V)

Time
0 5 10 15 20 25 30 35
0
10
20
30
40
50
60
10 15 20 25
5
4
3
1
3 4
5
2
2